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A total of 51 pentose utilizing lactic acid bacteria (LAB) were isolated from acid-forming bacteria in the midgut of healthy mature Eri silkworm using de Man, Rogosa and Sharpe (MRS) agar containing 10 g/L xylose (MRS-xylose) as the carbon source supplemented with 0.04% (<i>w</i>/<i>v</i>) bromocresol purple. Further analysis of 16S rRNA gene sequences revealed the highest prevalence of up to 35 enterococci isolates, which included 20 isolates of <i>Enterococcus mundtii</i>, followed by <i>Entercoccus faecalis</i> (eight isolates), <i>Weissella cibaria</i> (four isolates), <i>Enterococcus hirae</i> (two isolates), <i>Enterococcus lactis</i> (one isolate), and <i>Enterococcus faecium</i> (one isolate). All 51 LAB isolates showed positive growth on MRS containing a range of polysaccharides as the sole carbon source. All isolates were able to grow and form clear zones on MRS supplemented with 1 g/L xylose, while <i>E. faecalis</i> SC1, <i>E. faecalis</i> SCT2, and <i>E. hirae</i> SX2 showed tannin tolerance ability up to 5 g/L. Moreover, five isolates showed antimicrobial activity against Eri silkworm pathogens, including <i>Bacillus cereus</i>, <i>Staphylococcus aureus</i>, and <i>Proteus vulgaris</i>, with <i>E. hirae</i> SX2 having the highest inhibitory effect. Supplementation of live <i>E. hirae</i> SX2 on castor leaves significantly improved the weight and reduced the silkworm mortality when compared with the control group (<i>p</i> < 0.05). This cocci LAB can be considered as the new probiotic for Eri culture. Additionally, this finding presented the perspective of non-mulberry silkworm that could also be used as the model for further applying to new trends of the sericulture industry.