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The first part of the study aimed to isolate, characterize, and identify wild lactic acid bacteria (LAB) strains from alfalfa silage produced in a tropical area. LAB strains were isolated from alfalfa silage ensiled for 1, 3, 7, 14, 28, and 56 days (d) and were identified by sequencing the 16S rRNA gene. The second part aimed to investigate the effects of wild LAB strains on the nutritive and fermentative characteristics of alfalfa silage. This trial was conducted according to a completely randomized design in a 4 × 2 factorial scheme [four inoculants (I) × two harvests (H)], (n = 4). The inoculants were: (1) no inoculant (CTRL), (2) Lactobacillus pentosus (AV 14.17); (3) L. pentosus + Lactobacillus brevis + Pediococcus acidilactici (Combo); and (4) commercial inoculant (CI). Alfalfa forage (7 kg) was ensiled in 10 L buckets and opened after 90 d. Seventy-seven strains were isolated. Pediococcus, Lactobacillus, and Weissella represented 52.0, 24.7, and 20.8% of the isolates, respectively. For the first harvest, Combo, CI, and all inoculated silages showed lower acid detergent fiber ADF, neutral detergent fiber (NDF), and ammonia nitrogen (NH3-N), respectively. Silage fermented with AV14.17 presented greater residual water-soluble carbohydrate (WSC) in the second harvest and showed the lowest pH in both harvests. AV14.17 strain has potential as an inoculant for alfalfa silage production.