ABSTRACT: Mobile health (mHealth) technologies have been identified as promising strategies for improving access to healthcare delivery and patient outcomes. However, the extent of availability and use of mHealth among healthcare professionals in Ghana is not known. The study's main objective was to examine the availability and use of mHealth for disease diagnosis and treatment support by healthcare professionals in the Ashanti Region of Ghana. A cross-sectional survey was carried out among 285 healthcare professionals across 100 primary healthcare clinics in the Ashanti Region with an adopted survey tool. We obtained data on the participants' background, available health infrastructure, healthcare workforce competency, ownership of a mobile wireless device, usefulness of mHealth, ease of use of mHealth, user satisfaction, and behavioural intention to use mHealth. Descriptive statistics were conducted to characterise healthcare professionals' demographics and clinical features. Multivariate logistic regression analysis was performed to explore the influence of the demographic factors on the availability and use of mHealth for disease diagnosis and treatment support. STATA version 15 was used to complete all the statistical analyses. Out of the 285 healthcare professionals, 64.91% indicated that mHealth is available to them, while 35.08% have no access to mHealth. Of the 185 healthcare professionals who have access to mHealth, 98.4% are currently using mHealth to support healthcare delivery. Logistic regression model analysis significantly (p < 0.05) identified that factors such as the availability of mobile wireless devices, phone calls, text messages, and mobile apps are associated with HIV, TB, medication adherence, clinic appointments, and others. There is a significant association between the availability of mobile wireless devices, text messages, phone calls, mobile apps, and their use for disease diagnosis and treatment compliance from the chi-square test analysis. The findings demonstrate a low level of mHealth use for disease diagnosis and treatment support by healthcare professionals at rural clinics. We encourage policymakers to promote the implementation of mHealth in rural clinics.