In the clinical setting, surface disinfection is an important measure to reduce the risk of cross-transmission of microorganisms and the risk of nosocomial infections. Standardized methods can be used to evaluate said disinfection procedures, as well as the effectiveness of the active ingredients used for disinfection. However, despite standardization, the results of such methodologies are still determined by several factors and incorrect results may lead to invalid assumptions about the effectiveness of a disinfectant, posing significant health risks for patients and health personnel. The objective of this study was to evaluate several determinants for the recovery of Pseudomonas aeruginosa and other test organisms to establish their influence on the results of standardized disinfection methodologies and to find Gram-negative strains that could be used as suitable replacements of P. aeruginosa. The effects of inoculum application, drying times and temperatures as well as surface material on the survival and recovery of the test organisms were evaluated using Student-T test, one-way ANOVA and Tuckey's multiple comparison tests. Our results show that temperature and length of drying as well as application method and the material of germ carriers have significant effects on the recovery of P. aeruginosa cells and therefore influence the outcome of the methodologies used. This study also presents data showing that P. aeruginosa could be replaced with the Gram-negative species Acinetobacter baumannii, a test organism already in many standardized methodologies, which responds better under the same circumstances and has a behavior similar to that of P. aeruginosa in disinfectant efficacy tests.