This study assesses the abundance of fish in different habitats of Negombo Lagoon, a human-influenced, highly productive tropical lagoon in Sri Lanka, and the effects of physico-chemical parameters on their abundance. Six different habitats, i.e., three mangrove habitats, seagrass bed associated habitat, brushpile, and mud and sand flat were selected as sampling sites representing three major areas, Kadolkele, Dungalpitiya, and Kepungoda. Fish were sampled using a drag net with a stretch mesh size of 50 mm and salinity, Surface Water Temperature, Dissolved Oxygen, pH, Nitrate–N and Orthophosphate were measured in each habitat from January to December 2018 by making monthly field visit. A total of 45 fish species belonging to 30 families were identified. The highest species abundance was reported in seagrass associated habitat, while the lowest was in Kadolkele mangrove habitat (p < 0.05; ANOVA) due to its low level of DO (1.92 mg dm−3) and high Orthophosphate (1.02 mg dm−3) content. Different life stages of fish shared different habitats and small juveniles were abundant in association with seagrass bed (67.09%) while mature ones were dominant (42.12%) in brushpile area. This study proves that the abundance of fish varies with different physico-chemical parameters and, according to the generalized additive model, the highest abundance of fish is in association with > DO of 4 mg dm−3, low level of Orthophosphate (0.5–0.75 mg dm−3), 0.5–1.5 mg dm−3 of Nitrate–N, and 20–30 ppt salinity. The findings of this study are important to understand the spatial and seasonal patterns of fish abundance in a tropical lagoon and how physico-chemical parameters affect their abundance.