Further investigation into a fish gut-derived fungus Evlachovaea sp. CMB-F563, previously reported to produce the unprecedented Schiff base prolinimines A–B (1–2), revealed a new cryptic natural product, N-amino-l-proline methyl ester (5)—only the second reported natural occurrence of an N-amino-proline, and the first from a microbial source. To enable these investigations, we developed a highly sensitive analytical derivitization methodology, using 2,4-dinitrobenzaldehyde (2,4-DNB) to cause a rapid in situ transformation of 5 to the Schiff base 9, with the latter more readily detectable by UHPLC-DAD (400 nm) and HPLC-MS analyses. Moreover, we demonstrate that during cultivation 5 is retained in fungal mycelia, and it is only when solvent extraction disrupts mycelia that 5 is released to come in contact with the furans 7–8 (which are themselves produced by thermal transformation of carbohydrates during media autoclaving prior to fungal inoculation). Significantly, on contact, 5 undergoes a spontaneous condensation with 7–8 to yield the Schiff base prolinimines 1–2, respectively. Observations made during this study prompted us to reflect on what it is to be a natural product (i.e., 5), versus an artifact (i.e., 1–2), versus a media component (i.e., 7–8).