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This study aimed to determine the factors affecting the utilization of PMTCT services among mothers attendingthe ART clinic at Kiryandongo General Hospital. This was a facility-based cross-sectional which recruited a totalof 185 pregnant and lactating mothers attending Kiryandongo Hospital. STATA software version 14.0 was used fordata analysis. To identify factors affecting utilization of PMTCT services, univariate analysis and modified Poissonregression run and crude prevalence ratios with 95% confidence interval were used to determine the level ofsignificance at bivariate meanwhile adjusted prevalence ratios were calculated at multivariate analysis to establishindependent significant factors. The mean age of the study participants was 25.6 with a standard deviation of 6.Results showed that the mean score on knowledge was 5.21 with a standard deviation of 1.36. The median score was5 with an interquartile range of 4 to 6. The majority of 59.46% (110/185) of the study participants had inadequateknowledge of PMTCT meanwhile less than of the study participants 40.54% (75/185) had adequate knowledge. Thefactors independently affecting utilization of PMTCT services were; Unemployment (aPR 4.05, 95%CI 2.19-7.50,P<0.001), Rude and harsh attitude of health workers (aPR 2.17, 95%CI 1.38-3.44, P=0.001). Unsatisfactory services(Apr 2.40, 95%CI 1.22-4.74, P=0.011). The study has shown that there are more than half of the pregnant womenand lactating mothers attending Kiryandongo Hospital have inadequate knowledge of PMTCT services. Theproportion of mothers found with adequate knowledge of PMTCT in the present study is low compared to moststudies done in Africa. At Kiryandongo Hospital, the utilization of PMTCT services among pregnant and lactatingmothers was found to be slightly high. Employment status was the only socio-demographic factor affecting theutilization of PMTCT services meanwhile Rude and harsh attitudes of health workers and unsatisfactory serviceswere the health facility-related factors affecting the utilization of PMTCT services.Keywords: HIV, PMTCT services, Children, Pregnant mothers, Lactating mothers.