Healthcare professionals handling chemotherapy drugs are at occupational risk if proper precautions are not taken. Despite international recommendations for the safe handling of hazardous drugs, data on healthcare providers' knowledge, handling practices, and challenges in Libya are limited. The study aimed to assess the knowledge and safe handling practices of healthcare providers regarding chemotherapy in two hospitals in Benghazi, Libya, and identify factors associated with safe handling practices. A cross-sectional study was conducted between October 2024 and August 2025 among pharmacists and nurses at Benghazi Medical Centre and Children's Hospital. Participants completed a questionnaire on demographics, knowledge, handling practices, and workplace challenges. Non-parametric tests and Cronbach's alpha were used to analyse the data and assess questionnaire reliability. A total of 56 healthcare workers participated. Respondents demonstrated partial understanding of chemotherapy-related concepts, with 21.4% achieving good knowledge, and over one-third demonstrating poor knowledge. Pharmacists scored significantly higher than nurses (p = 0.001). Half of the participants reported safe handling practices, and previous chemotherapy safety training was significantly associated with higher practice scores (p = 0.022). Participants showed good knowledge of general chemotherapy principles but limited knowledge of oral chemotherapy and protective glove use. The main challenges identified were heavy workload (85.7%), lack of management support (66.1%), and inadequate chemotherapy training (64.3%). The knowledge and practice scales demonstrated modest (Cronbach’s α = 0.548) and good (Cronbach’s α = 0.717) reliability, respectively. Healthcare providers demonstrated acceptable handling practices but insufficient knowledge of chemotherapy safety and reported limited organisational support. Regular training, stronger institutional support, and clear policies are needed to improve the safe handling of chemotherapy in Libya.