Surface of Lactobacillus crispatus DSM 20584 (LBC) and Lactobacillus rhamnosus GG (LGG) from stationary and exponential phase were chemically modified using hexanoic anhydride (HA) and octanal via grafting hydrophobic moieties onto the bacterial surface hydroxyl and amine groups. The physicochemical properties of the bacteria were measured using a range of surface-sensitive methods including x-ray photoelectron spectroscopy (XPS), zeta potential measurement, contact angle measurement (CAM) and microbial adhesion to solvents (MATS). Before modification, the surface of two strains was distinctly different, where LBC was covered by hydrophobic surface-layer proteins (SLPs) while LGG was hydrophilic with the rich presence of polysaccharides. Surface hydrophilic polymers rendered steric hindrance of LGG against autoaggregation, whereas LBC lacking polysaccharides showed strong autoaggregation. After HA and octanal modifications, the intrinsic surface differences between two strains were reduced according to the Principal Component Analysis (PCA). The enhancement of hydrophobicity by HA and octanal was most likely derived from the lowered Lewis acid-base characters via elimination of hydroxyl and amine groups. Chemical modification using the two treatments can be a useful tool to tune the surface of lactic acid bacteria, which might be further applied to other microorganisms, enabling applications such as altered bacterial adhesive behaviors and biofilm formation.