Probiotics are defined as live organisms in the host that contribute to health benefits. Lactobacillus gasseri LM1065, isolated from human breast milk, was investigated for its probiotic properties based on its genome. Human breast milk was donated by a healthy woman. The isolated Lactobacillus strain was identified as L. gasseri and named LM1065. Complete genome sequencing and de novo assembly were performed using the PacBio RS II system and hierarchical genome assembly process (HGAP). Probiotic properties were determined by the resistance of the strain to gastric conditions, adherence ability, enzyme production, and safety assessment. The fungistatic effect and the ability to inhibit hyphae transition were studied using the cell-free supernatant (CFS) of L. gasseri LM1065. L. gasseri LM1065 showed high gastric pepsin tolerance and mild tolerance to bile salts. Auto-aggregation ability and hydrophobicity were measured to be 61.21% and 61.55%, respectively. These properties resulted in the adherence of the bacteria to the human intestinal epithelial cells (2.02%). In addition, antibiotic-resistance genes and putative virulence genes were not predicted in the complete genome sequence of L. gasseri LM1065, and antibiotic susceptibility was satisfied by the criteria of the European Food Safety Authority using a cut-off test. CFS showed a fungistatic effect and suppressed the tricarboxylic acid cycle in Candida albicans (29.02%). CFS also inhibited the transition to true hyphae and damaged the blastoconidia. This study demonstrates the essential properties of this novel probiotic, L. gasseri LM1065, and its potential to inhibit vaginal Candida albicans infection.