Introduction: The safe handling and administration of chemotherapeutic drugs require specialized knowledge and skills to ensure patient safety and minimize occupational risks. Despite global guidelines, context-specific evidence on nurses’ competencies in tertiary health facilities in Benin City remains limited. This study aimed to assess nurses’ knowledge and practices regarding safe chemotherapy handling and administration. Methods: A descriptive cross-sectional study was conducted among 278 randomly selected nurses in a tertiary hospital. Data were collected using a validated structured questionnaire. Descriptive statistics summarized the data, and multiple logistic regression was used to examine associations at a 5% significance level using IBM SPSS version 28.0. Results: Among participants, 17% had poor knowledge, 63% had fair knowledge, and only 20% demonstrated good knowledge. Regarding practice, 51% exhibited poor practice. Key factors supporting safe chemotherapy handling included training and certification (mean = 3.94), incident encounters (mean = 3.73), protocol adherence (mean = 2.93), and use of personal protective equipment (mean = 2.82). Logistic regression analysis showed that being female (OR = 4.90; 95% CI: 0.23–3.54; p = 0.479), aged 18–34 years (OR = 7.03; 95% CI: 0.06–8.28; p = 0.298), and holding a bachelor’s degree (OR = 7.69; 95% CI: 0.05–7.10; p = 0.174) were associated with higher odds of better practice, although these were not statistically significant. Conclusions: Nurses demonstrated moderate knowledge but suboptimal practices in chemotherapy handling, indicating critical competency gaps. Strengthening training, protocol adherence, and institutional policies is essential to improve safe chemotherapy administration.