Two of the important contamination sources by antineoplastic drugs (AD) are hospitals and pharmacies. Unwanted releases have been documented during all steps of the preparation and administration of these hazardous drugs to patients leading to contamination of both working places and outside environment (transfer by aerosols, contaminated materials, or water after cleaning). Here we present results of a long-term project from 21 hospitals in the Czech Republic (971 samples; 2008–2016) investigating the contamination by major AD-5-fluorouracil (FU), cyclophosphamide (CP) and platinum drugs (total Pt as a sum of broadly used Pt-based drugs cisplatin, oxaliplatin, and carboplatin). In general, lower median levels of contamination have been found in pharmacies, which could be attributed to personnel education and higher safety working standards. On the other hand, surface contamination in other hospital areas exceeded the suggested threshold guidance values (TGVs) in up to 40% of samples depending on the monitored drug (TGVs being 67, 12, and 38 pg per square cm for CP, Pt, and FU, respectively). The highest values, maxima exceeding 29,000 and 49,000 pg per square cm for CP and FU, respectively, have repeatedly been found in outpatient clinics. The monitoring and discussions with responsible managers promoted the implementation of proper procedures and technologies that resulted in an overall decrease of the contamination during the monitored period.