Mules found working in Egyptian brick kilns are often faced with poor welfare from being over worked, overloaded, having multiple lesions from ill-fitted harnesses, poor body scores, and receiving aversive treatment by handlers. Reports have frequently revealed aggressive responses by mules toward their handlers. The main goal of this study was to investigate whether mule aggression is an innate act toward people or is a reactive response to rough handling procedures by their handlers. A total of 374 mules from 50 different kilns were assessed and their handlers were interviewed. The handler's questionnaire recorded the following parameters: handler's age, experience, and common beliefs about the aggression of brick kiln mules, and also data regarding load weights, working hours, and husbandry procedures carried out by the handlers were collected. The data were analyzed and correlations between parameters were tested using SPSS 17.1. Handlers' data showed that 79% of participants believed that mules are inherently difficult to handle, 65% used nose ropes/metal chain for driving their mules, and 67% responded that mules must be beaten to work properly. Behavioral assessment revealed that 66% of mules were alert. Approach tests indicated that 30% of the mules exhibited signs of aggression (e.g., bite threat) when approached by an unfamiliar handler and only 16% showed signs toward their handlers (familiar). The assessment of body lesions showed that mistreatment-induced lesions (42%) were more predominant than other categories of body lesions. Significant correlations (P ≤ 0.05) were found between mules' aggressive responses toward observers and the following parameters; body condition score (rs = 0.42), along with the handler's age (rs = −0.53), level of experience (rs = −0.34), handler's common beliefs about mule aggression (rs = 0.64), and the nature of the work they were involved with at the kilns (work hours, rs = −0.63; load weight, rs = 0.38). Based on the results of this study, we concluded aggressive interactions exhibited by mules were most likely initiated by harsh, violent handling.