In the present review, we briefly summarize the biotechnological applications of microbial β-xylosidases in the processing of agro-industrial residues into fuels and chemicals and report the importance of using immobilization techniques to study the enzyme. The advantages of utilizing genes that encode β-xylosidases are readily apparent in the bioconversion of abundant, inexpensive, and renewable resources into economically important products, such as xylitol and bioethanol. We highlight recent research characterizing fungal and bacterial β-xylosidases, including the use of classical biochemical methods such as purification, heterologous recombinant protein expression, and metagenomic approaches to discovery β-xylosidases, with focus on enzyme molecular and kinetic properties. In addition, we discuss the relevance of using experimental design optimization methodologies to increase the efficacy of these enzymes for use with residual biomass. Finally, we emphasize more extensively the advances in the regulatory mechanisms governing β-xylosidase gene expression and xylose metabolism in gram-negative and gram-positive bacteria and fungi. Unlike previous reviews, this revision covers recent research concerning the various features of bacterial and fungal β-xylosidases with a greater emphasis on their biochemical characteristics and how the genes that encode these enzymes can be better exploited to obtain products of biotechnological interest via the application of different technical approaches.