tRNAs purified from non-pathogenic Escherichia coli strains (NPECSs) possess cytotoxic properties on colorectal cancer cells. In the present study, the bioactivity of tRNA halves and tRNA fragments (tRFs) derived from NPECSs are investigated for their anticancer potential. Both the tRNA halves and tRF mimics studied exhibited significant cytotoxicity on colorectal cancer cells, with the latter being more effective, suggesting that tRFs may be important contributors to the bioactivities of tRNAs derived from the gut microbiota. Through high-throughput screening, the EC83 mimic, a double-strand RNA with a 22-nucleotide (nt) 59-tRF derived from tRNA-Leu(CAA) as an antisense chain, was identified as the one with the highest potency (50% inhibitory concentration [IC50] = 52 nM). Structure-activity investigations revealed that 29-O-methylation of the ribose of guanosine (Gm) may enhance the cytotoxic effects of the EC83 mimic via increasing the stability of its tertiary structure, which is consistent with the results of in vivo investigations showing that the EC83-M2 mimic (Gm modified) exhibited stronger antitumor activity against both HCT-8 and LoVo xenografts. Consistently, 4-thiouridine modification does not. This provides the first evidence that the bioactivity of tRF mimics would be impacted by chemical modifications. Furthermore, the present study provides the first evidence to suggest that novel tRNA fragments derived from the gut microbiota may possess anticancer properties and have the potential to be potent and selective therapeutic molecules.