•AdV pollution in fishing ports was associated with nearby polluted river water.•HAdV 41 and PAdV 5 were the dominant serotypes in the waters of the fishing ports.•The seasonal distributions of HAdV and PAdV differed significantly.•The AdV detection rate significantly differed according to regional distribution.•The presence of AdV was associated with total plate count, pH, and salinity. Adenoviruses (AdVs) are a major cause of clinical infections and have been proposed as indicators of water quality. However, quantitative data on the environmental prevalence of AdVs is lacking. We investigated the prevalence, distribution, seasonal occurrence, quantity, and genotype of AdVs in 13 fishing harbours in Taiwan. AdVs in the water samples were isolated by membrane filtration and the AdV DNA was extracted. Next, AdVs were detected using nested polymerase chain reaction. Genotyping and phylogenetic analysis were performed to identify various AdV genotypes present in the water samples. The F species human AdV (HAdV) serotype 41 (63.6%) and C species porcine AdV (PAdV) serotype 5 (33.3%) were more prevalent than the other serotypes. The prevalence of AdVs was highest in the fall, followed by in the spring and summer. Among the fishing harbours, the highest detection rate of AdVs was observed in Yenpudongang in all seasons. However, Puoziliao was the only site at which AdVs were not detected during the study period. AdV detection at sampling sites may be correlated with sewage and livestock wastewater discharge via outflow of nearby rivers to fishing ports. Statistical analysis (Mann–Whitney U test) based on data from water quality indicators revealed that the presence of AdVs was significantly associated with the heterotrophic plate count, pH, and salinity. Human and swine population data from nearby local townships and river/drainage basins were collected from the Taiwan Central Government's website. The data were analyzed using Spearman's rank correlation coefficient to determine the relationship between the prevalence of AdVs, HAdVs, and PAdVs in fishing harbours, and microbial water quality indicators. Statistical evidence indicated that the detection levels of HAdVs and PAdVs in fishing harbours were mainly associated with human and swine populations in the corresponding river/drainage basin, respectively. Additionally, the swine population in the river/drainage basin was positively correlated with microbial water quality indicators. Display omitted