Our objectives were to identify the prevalence of negligence of laboratory animals in Brazil, to determine the primary factors associated with its occurrence and to suggest prevention strategies. A questionnaire was made available online between October 2015 and March 2016. A total of 116 respondents with experience in the use of laboratory animals and/or the use of alternative methods answered the questionnaire. Most respondents were women (77 respondents, 66.4%), a significant proportion had a degree in Veterinary Medicine (31 respondents, 27.2%), and a majority used animals in their work (88 respondents, 75.9%). Of the 88 animal users, 23 supplied information on the numbers and species of animals they used. When asked whether they knew that Brazilian law forbade animal experimentation when alternative methods exist, seven (9.1%) respondents mentioned Act 9605/1998. Most, but not all, respondents (96 respondents, 82.8%) submitted their projects to an Animal Use and Ethics Committee (AUEC), and many (65 respondents, 56%) reported their belief that animal neglect occurred at their institution. Negligence was found to be associated with: institutions where the numbers of animals used were not recorded ( p = 0.008); institutions where respondents were unaware of the relevant legislation, that is, Act 9605/1998 ( p = 0.042); or where there was evidence that not all project proposals were submitted to the AUEC or evidence of no submissions at all ( p = 0.022). Negligence of animals was found to be highly prevalent. Prevention strategies might involve increased transparency to the general public, the empowerment of individuals that work with animals to report any concerns, optimised inspection of facilities where animal work is carried out and significant improvements to the role of AUECs.