Late initiation of antiretroviral therapy (ART) is associated with worse health outcomes for people living with HIV. In 2019, Cambodia implemented a same-day policy allowing people with HIV to start ART on the day they were diagnosed. Using case data collected by Cambodia’s National Center for HIV/AIDS Dermatology and STDs, this study examined trends and factors influencing late ART initiation among newly diagnosed HIV cases attending three clinics in Kampong Thom province from 2014 to 2023. Factors linked to late ART initiation (defined as starting treatment >1 day after diagnosis) were assessed using descriptive statistics and logistic regression. Statistical significance was set at P < 0.05. The study included 741 newly diagnosed HIV cases, with a mean age of 45.7 years at diagnosis (standard deviation: 10.2); 57.1% (423) of cases were female and more than 70% (504/704) were employed. In the 5-year period before the implementation of same-day ART initiation (2014–2018), 91% (325/356) of cases had late ART initiation. In the subsequent 5-year period (2019–2023), the proportion of cases with late ART initiation averaged 23%, and in 2023 was just 3%. Late ART initiation was more common at the Baray Santuk clinic (adjusted odds ratio: 3.94, 95% confidence interval: 1.99–7.81, P < 0.001), likely due to a lack of staff dedicated to HIV care. The findings demonstrate that same-day ART initiation is feasible in resource-limited settings and that adequate staffing can further improve prompt ART initiation.