The main objectives of our research were the characterization of Staphylococcus epidermidis strains isolated from cleanrooms and human S. epidermidis strains, and study of their adaptation to didecyldimethylammonium chloride (DDAC)-based disinfectant and to DDAC itself. The susceptibilities to the disinfectant of 57 S. epidermidis strains isolated from cleanrooms were determined according to their minimum inhibitory concentrations (MICs), which ranged from 1.3 to 40.8 mg/L for the disinfectant, and from 0.1 to 4.5 mg/L for DDAC. In 15 clinical strains, the MICs were 10.2 mg/L for the disinfectant and 0.1 mg/L for DDAC. The qacA/B and qacC genes were present in the majority of cleanroom strains (96.5%), with almost half showing resistance to gentamicin (47.4%), but with low cefoxitin resistance (3.5%); 40.4% of these cleanroom strains were biofilm formers. Seven S. epidermidis strains were exposed to gradually increased concentrations of disinfectant or DDAC. Adaptation to the disinfectant was not seen, but for DDAC, 4 strains became adapted and 3 strains became resistant. Their corresponding MICs increased from 2- to 180-fold. Compared to their non-adapted counterparts, these adapted/resistant strains became cross-resistant to benzalconium chloride (3/7) and antibiotics (4/7), showed smaller cell size (6/7), altered their fatty-acid profiles (7/7), increased their efflux pump activities (5/7), and became stronger biofilm formers (3/7). Analysis for differentially expressed genes of an adapted (Se11Ad) and a resistant (Se18To) strain using RNA-seq analysis with criterium of fold change (FC) > 5 and statistical significance (p) < 0.05, revealed several adaptation mechanisms. Both strains had up-regulated genes for efflux pumps (e.g., arsenic efflux pump) and transport systems (e.g., amino acids, peptides, phosphate ions, nucleotides transport). For Se11Ad, down-regulation of the Agr system was seen, which results in stronger biofilm formation, as an important adaptation mechanism. For Se18To, several genes involved in cell-wall synthesis were down-regulated, which resulted in thickening of the cell wall. S. epidermidis studies and knowledge of their adaptation and resistance development mechanisms are crucial for the definition of effective disinfection strategies.