The church consists of a single nave and chancel with a small western tower projecting into the nave. Except for the four Early English windows (two on the north wall and two on the south) and a Decorated window over the font, the building is Perpendicular in style. The medieval glass was destroyed by the parliamentary forces in 1644 during the civil War.
The Norman west front includes a round-headed doorway in the centre of an arcade formerly containing stone seats known 'penniless benches'. Originally there were five lancet windows in the second storey of the tower, but four were cut away to make room for a larger Perpendicular window. A tablet by the south arcade commemorates St. Edmund of Abingdon, who was contemporary with the earliest parts of the church. Inside the porch is a rare stone lantern, with a stone funnel.
The church is unusual in that the nave crosses the course of the River Stert. This river, which now flows in a culvert under Stert Street, marked the western boundary of the Parish of St. Nicolas', and so the tower and part of the nave were outside the parish. An arch over the river can be seen in the north wall from outside, and the water can be heard through a grating in the road on the south side.
On the north wall, halfway up the nave, is a stone cross uncovered during the 1881 restoration from beneath a layer of plaster. The figure of Christ which it once bore was probably removed during the civil War. Above the cross, to the left, is the attractive monument to Walter Dayrell, the first regularly appointed Recorder of the Borough.
Further east, is a quasi-transept containing the tomb of John Blacknall, a fine monument dating from 1684. John Blacknall was the grandson of the purchaser of the Abbey after its dissolution, and by his will he augmented the funds for supporting the readers at St. Nicolas' as well as providing for a weekly distribution of bread to the poor.
The pulpit, on the south wall of the nave, is Jacobean but has lost two storeys of its height. Behind is a small Early English window, partly blocked when the Abbey Gateway was rebuilt in the 15th century.
In the Chancel is a tablet on the south wall which was erected in memory of Richard Bowles, rector from 1775 to his death in 1804 who left £2,000 for a Fellow of Trinity College, Oxford, to preach a sermon every Sunday.
On the south wall of the clergy vestry is a fragment of an early reredos found under the panelling during the 1881 restoration. It depicts the Virgin Mary and St. John on either side and St. Nicolas in Bishop's vestments.
When the chancel was rebuilt after the fire of 1953 the Arms of Abingdon Abbey and of Trinity College, Oxford, were placed on the ends of the hammer beams together with those of four benefactors - Peter Heylin, a Sub Dean of Westminster Abbey who died in 1661, and largely due to whose efforts the Church was not demolished during the Commonwealth, Walter Dayrell who died in 1628, John Blacknall, and John Roysse who died in 1571.
The organ, built by Nicholson, dates from after the fire in 1953. The bells contain members of a peal of six cast by Abel Rundal of Gloucester in 1741.
Some historical photographs of the church are available from English Heritage.