Background Malaria remains a major public health threat accounting for 30.4 % of disease morbidity in outpatient clinic visits across all age groups in Uganda. Consequently, malaria control remains a major public health priority in endemic countries such as Uganda. Experiences from other countries in Africa that revised their malaria case management suggest that health workers adherence may be problematic. Methods A descriptive, cross-sectional design was used and collected information on health system, health workers and patients. Using log-binomial regression model, adjusted prevalence risk ratios (PRRs) and their associated 95 % confidence intervals were determined in line with adherence to new treatment guidelines of parasitological diagnosis and prompt treatment with artemisinin combination therapy (ACT). Results Nine health centres, 24 health workers and 240 patient consultations were evaluated. Overall adherence to national malaria treatment guidelines (NMTG) was 50.6 % (122/241). It was significantly high at HC III [115 (53 %)] than at HC IV (29 %) [PRR = 0.28 (95 % CI 0.148 0.52), p = 0.000]. Compared to the nursing aide, the adherence level was 1.57 times higher among enrolled nurses (p = 0.004) and 1.68 times higher among nursing officers, p = 0.238, with statistical significance among the former. No attendance of facility malaria-specific continuing medical education (CME) sessions [PRR = 1.9 (95 % CI 1.29 2.78), p = 0.001] and no display of malaria treatment job aides in consultation rooms [PRR = 0.64 (95 % CI 0.4 1.03), p = 0.07] was associated with increased adherence to guidelines with the former showing a statistical significance and the association of the latter borderline statistical significance. The adherence was higher when the laboratory was functional [PRR = 0.47 (95 % CI 0.35 0.63)] when the laboratory was functional in previous 6 months. Age of health worker, duration of employment, supervision, educational level, and age of patient were found not associated with adherence to new treatment guidelines. Conclusion Adherence to malaria treatment guidelines in Uganda is sub-optimal. There is an urgent need for deliberate interventions to improve adherence to these guidelines. Possible interventions to be explored should include: provision of job aides and improved access to laboratory services. There is also a need for continuous medical educational sessions for health workers, especially those at higher-level facilities and higher cadres, on adherence to guidelines in management of fever, including management of other causes of fever.