Background Mobile phones are widely used in clinical settings and could be colonized by potential pathogenic bacteria which may lead to hospital-acquired infections (HAIs) transmission. This study aimed to determine the prevalence of bacterial contamination of healthcare workers’ (HCWs) mobile phones, identify bacterial isolates, and assess the factors associated with mobile phone contamination. Methods Self-administered questionnaire was used to collect the information on the demographic characteristics and the use of mobile phones. A total of 111 HCWs’ hands and their mobile phones were swabbed, then bacterial culture, isolation, and identification were performed. Univariate and multivariable logistic regression were applied to identify factors associated with mobile phone bacterial contamination. Results Totally 106 (95.5%) of the 111 mobile phones investigated were contaminated with bacteria. Staphylococcus epidermidis (13/111), Acinetobacter baumannii (4/111) and Staphylococcus aureus (3/111) were the predominant bacterial isolates from HCWs’ mobile phones. Univariate analyses showed that age, gender, profession and the frequency of mobile phone utilization were significantly associated with the number of bacterial colonization. Frequency of phone utilization (OR 8.366; 95% CI 1.496–46.797) was found to be the most significant factors associated with the qualified rate of mobile phones bacterial load. In addition, phone cover using was associated with the increased risk of mobile phone bacterial contamination. Conclusion There was cross-contamination between hands and phones. It is necessary to develop guidelines for mobile phone cleaning. Special attention needs to be paid to the disinfection of mobile phone covers to reduce contamination and transmission of pathogens.