Non-specific lipid transfer proteins (nsLTPs) stand out among plant-specific peptide superfamilies due to their multifaceted roles in plant molecular physiology and development, including their protective functions against pathogens. These antimicrobial agents have demonstrated remarkable efficacy against bacterial and fungal pathogens. The discovery of plant-originated, cysteine-rich antimicrobial peptides such as nsLTPs has paved the way for exploring the mentioned organisms as potential biofactories for synthesizing antimicrobial compounds. Recently, nsLTPs have been the focus of a plethora of research and reviews, providing a functional overview of their potential activity. The present work compiles relevant information on nsLTP omics and evolution, and it adds meta-analysis of nsLTPs, including: (1) genome-wide mining in 12 plant genomes not studied before; (2) latest common ancestor analysis (LCA) and expansion mechanisms; (3) structural proteomics, scrutinizing nsLTPs’ three-dimensional structure/physicochemical characteristics in the context of nsLTP classification; and (4) broad nsLTP spatiotemporal transcriptional analysis using soybean as a study case. Combining a critical review with original results, we aim to integrate high-quality information in a single source to clarify unexplored aspects of this important gene/peptide family.