This study aimed to identify features of the road environment that increased the risk of on-road bicycle crashes in Perth, Australia between 2014-2017. This case-control study used a combination of an in-depth crash study and naturalistic study to compare the road environment characteristics of 100 case (crash) sites and 300 control sites where no crash occurred using conditional logistic regression. For intersection sites, roundabouts (adjusted OR: 2.98, 95% CI: 1.18, 7.56) and traffic lights (adjusted OR: 3.86, 95% CI 1.29, 11.63) significantly increased the risk of a bicycle crash, compared to priority control/uncontrolled intersections. For midblock (non-intersection) sites, roads with an incline (upwards/downwards) significantly increased the risk of a crash (adjusted OR: 3.39, 95% CI: 1.02, 11.22), compared to level roads. This study highlighted the risk of roundabouts, traffic lights and roads with an incline for bicycle crashes. Treatments that reduce vehicle speeds and encourage cyclists to claim the lane at roundabouts, as well as careful road design and road maintenance at traffic lights, may reduce the risk of crashes for cyclists. While it is impossible to remove hills and slopes from the topography, it is possible to select routes to target for bicycle infrastructure which are predominantly level.