The rise of infectious diseases as a public health concern has necessitated the development of rapid and precise diagnostic methods. Imaging techniques like nuclear and optical imaging provide the ability to diagnose infectious diseases within the body, eliminating delays caused by sampling and pre-enrichments of clinical samples and offering spatial information that can aid in a more informed diagnosis. Traditional molecular probes are typically created to image infected tissue without accurately identifying the pathogen. In contrast, oligonucleotides can be tailored to target specific RNA sequences, allowing for the identification of pathogens, and even generating antibiotic susceptibility profiles by focusing on drug resistance genes. Despite the benefits that nucleic acid mimics (NAMs) have provided in terms of stabilizing oligonucleotides, the inadequate delivery of these relatively large molecules into the cytoplasm of bacteria remains a challenge for widespread use of this technology. This review summarizes the key advancements in the field of oligonucleotide probes for in vivo imaging, highlighting the most promising delivery systems described in the literature for developing optical imaging through in vivo hybridization.