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Global population declines in herpetofauna have been documented extensively. Southern Florida, USA, is an especially vulnerable region because of high impacts from land development, associated hydrological alterations, and invasive exotic species. To ask whether certain snake species have decreased in abundance over recent decades, we performed a baseline road survey in 1993-1994 in a rural area of southwest Florida (Lee County) and repeated it 21 y later (2014-2015). We sampled a road survey route (17.5 km) for snakes by bicycle an average of 1.3 times a week (n = 45 surveys) from June 1993 through January 1994 and 1.7 times a week from June 2014 through January 2015 (n = 61 surveys). Snake mortality increased significantly after 21 y, but this result may be due to increased road traffic rather than expanding snake populations. The snake samples were highly dissimilar in the two periods, suggesting changes in species composition. For example, one species showed a highly significant decrease in abundance (Rough Greensnake, Opheodrys aestivus) while another showed substantial increases (Ring-necked Snake, Diadophis punctatus). Because of uncertain differences in traffic volume between 1993 and 2015, other species offered ambiguous results in their abundance trends. Nevertheless, four additional species contributed at least 12.1% dissimilarity (North American Racer, Coluber constrictor; Red Cornsnake, Pantherophis guttatus; Eastern Ribbonsnake, Thamnophis saurita; and Southern Watersnake, Nerodia fasciata). Increases in human land-use due to development and agriculture along with associated loss of wetlands and native habitats may have contributed to the changes we documented. Future work should seek to understand fully these causes and the conservation needs of declining species.