The aim of this study was to improve the acid tolerance of Lactobacillus acidophilus by combining atmospheric and room temperature plasma (ARTP) mutation with adaptive laboratory evolution (ALE). To achieve a high mutation efficiency, 60 s was determined as the ideal exposure time for ARTP mutation of L. acidophilus with a survival rate of 5.91%. The ARTP-ALE mutant strain LAartp-ale2 displayed increased lactic acid stress tolerance with survival rates of 75.67% and 25.78% when cultured in pH 3.0 and 2.5, respectively, for 3 h. Physiological analysis revealed that the ARTP-ALE mutant exhibited a lower inner membrane permeability than that of the parental strain during acid stress. Furthermore, the mutant LAartp-ale2 produced more biofilm in response to lactic acid-induced acid stress and showed an increased hydrophobicity (87.2%) when compared to the parent strain (76.2%) at pH 2.5. LAartp-ale2 exhibited a higher unsaturated fatty acid (UFA) to saturated fatty acid (SFA) ratio that affected the physical state of the cell membrane for increased survival in pH 3.0 and 2.5. The mutation with ARTP coupled with ALE in the present study proved to be effective in enhancing the acid tolerance of L. acidophilus for potential industrial use.