It is widely recognized that subclinical mastitis (SCM) is an extensive problem in the dairy industry worldwide. It is of particular concern in developing countries. The aim of this study was to establish the prevalence of SCM in dairy cattle in the urban and peri-urban areas of Kampala, Uganda and to gain information about pathogens and antibiotic resistance patterns. The study was conducted as a field study in 18 smallholder dairy farms in peri-urban Kampala, Uganda. All cows at the farms were physically examined, and cows with signs of clinical mastitis were excluded. Cows (n = 195) were tested with California Mastitis Test (CMT), and udder quarters with CMT score ≥3 (scale 1–5) were milk sampled for bacteriological analysis. To allow further sub-analysis of the results, the stage of lactation, parity, milk production, production type, udder hygiene, and cow breed were recorded. Results indicate that 86.2 % (n = 168) of the tested cows had SCM in one or more quarters. The most common bacteriological outcome was infection with coagulase-negative staphylococci (54.7 %), followed by negative growth (24.9 %) and streptococci (16.2 %); all of which (n = 34) were sensitive to penicillin. Of the tested staphylococci (n = 17), the majority (58.9 %) were positive for penicillinase production. Factors with significant impact on the prevalence of SCM at cow level were the stage of lactation, parity, and production type. The results suggest that the prevalence of SCM in Uganda is substantially higher than reported in previous studies and in other comparable developing countries. This implies that SCM deserves more attention and that improvement in dairy cow husbandry in terms of hygiene and management is necessary.