Background Small non-coding RNAs play essential roles in gene regulation, however, the interplay between RNA groups, their expression levels and deregulations in tumorigenesis requires additional exploration. In particular, a comprehensive analysis of microRNA (miRNA), PIWI-interacting RNAs (piRNAs), and tRNA-derived small RNAs in human testis and testicular germ cell tumor (TGCT) is lacking. Results We performed small RNA sequencing on 22 human TGCT samples from 5 histological subtypes, 3 carcinoma in situ, and 12 normal testis samples. miRNA was the most common group among the sequences 18–24 nt in length and showed histology-specific expression. In normal samples, most sequences 25–31 nucleotides in length displayed piRNA characteristics, whereas a large proportion of the sequences 32–36 nt length was derived from tRNAs. Expression analyses of the piRNA population demonstrated global loss in all TGCT subtypes compared to normal testis. In addition, three 5′ small tRNA fragments and 23 miRNAs showed significant (p < 10−6) differential expression in cancer vs normal samples. Conclusions We have documented significant changes in the small RNA populations in normal adult testicular tissue and TGCT samples. Although components of the same pathways might be involved in miRNA, piRNA and tRNA-derived small RNA biogenesis, our results showed that the response to the carcinogenic process differs between these pathways, suggesting independent regulation of their biogenesis. Overall, the small RNA deregulation in TGCT provides new insight into the small RNA interplay.