Objective: This study aimed to evaluate two cleaning solutions for the chemical decontamination of antineoplastic agents on the surfaces of two biosafety cabinets routinely used for chemotherapy preparation in a hospital pharmacy. Methods: For almost 1 year (49 weeks), two different solutions were used for the weekly cleaning of two biosafety cabinets in a hospital pharmacy's centralized cytotoxic preparation unit. The solutions evaluated were a commercial solution of isopropyl alcohol (IPA) and water (70:30, vol:vol), and a detergent solution constituted by 10–2M of sodium dodecyl sulfate (SDS) with 20% IPA. Seven areas in each biosafety cabinet were wiped 14 times throughout the year, before and after the weekly cleaning process, according to a validated procedure. Samples were analyzed using a validated method of high-performance liquid chromatography coupled to mass spectrometry. The decontamination efficacy of these two solutions was tested for 10 antineoplastic agents: cytarabine, gemcitabine, methotrexate, etoposide phosphate, irinotecan, cyclophosphamide, ifosfamide, doxorubicin, epirubicin, and vincristine. Results: Overall decontamination efficacies observed were 82±6% and 49±11% for SDS solution and IPA, respectively. Higher contamination levels were distributed on areas frequently touched by the pharmacy technicians—such as sleeves and airlock handles—than on scale plates, gravimetric control hardware, and work benches. Detected contaminations of cyclophosphamide, ifosfamide, gemcitabine, and cytarabine were higher than those of the others agents. SDS solution was almost 20% more efficient than IPA on eight of the antineoplastic agents. Conclusion: Both cleaning solutions were able to reduce contamination levels in the biosafety cabinets. The efficacy of the solution containing an anionic detergent agent (SDS) was shown to be generally higher than that of IPA and, after the SDS cleaning procedure, biosafety cabinets demonstrated acceptable contamination levels.