This paper aimed to assess the success of cleaning and disinfection on microbiological contamination of anesthetic masks, which were used for automated isoflurane anesthesia for surgical castration of male piglets. Data collection took place on 11 farms in Southern Germany between September 2020 and June 2022. Each farm was visited three times (one farm having two different anesthesia devices was visited six times), and microbiological assessments took place at four sample points (SP): after unpacking the masks (SP0), after disinfection before anesthesia (SP1), after anesthesia of all piglets to be castrated in this run (SP2), and after disinfection after anesthesia (SP3). The microbiological assessment included the determination of total bacteria count, total count of hemolytic and non-hemolytic mesophilic aerotolerant bacteria and a qualitative detection of indicator bacteriaEscherichia (E.) coli, extended-spectrum beta-lactamase-producingE. coli(ESBL) and methicillin-resistantStaphylococcus aureus(MRSA). For analysis, a generalized linear mixed model was applied using farms and farm visits as random effects and sampling points nested in farm visits as fixed effect. The fixed effect was highly significant for all three variables (total bacteria count, total count of hemolytic and non-hemolytic mesophilic aerotolerant bacteria) (p < 0.001). The bacterial counts at SP0 were about the same as at SP3. Concerning indicator bacteria, their presence was highest at SP2 and lowest at SP3. No indicator bacteria were present at SP1. It can be concluded that disinfection of anesthetic masks, especially before performing anesthesia, may effectively protect piglets of the following batch against unwanted transmission of pathogens. These findings will help farmers plan cleaning and disinfection activities.