Lettuce (Lactuca sativaL., Asteraceae) is a popular vegetable leafy crop playing a relevant role in human nutrition. Nowadays, novel strategies are required to sustainably support plant growth and elicit the biosynthesis of bioactive molecules with functional roles in crops including lettuce. In this work, the polyphenolic profile of lettuce treated with glutamic acid (GA), humic acid (HA), and their combination (GA + HA) was investigated using an untargeted metabolomics phenolic profiling approach based on high-resolution mass spectrometry. Both aerial and root organ parts were considered, and a broad and diverse phenolic profile could be highlighted. The phenolic profile included flavonoids (anthocyanins, flavones, flavanols, and flavonols), phenolic acids (both hydroxycinnamics and hydroxybenzoics), low molecular weight phenolics (tyrosol equivalents), lignans and stilbenes. Overall, GA and HA treatments significantly modulated the biosynthesis of flavanols, lignans, low molecular weight phenolics, phenolic acids, and stilbene. Thereafter, antioxidant capacity was evaluated in vitro with 2,2-diphenyln-1-picrylhydrazyl (DPPH), 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and cupric ion reducing antioxidant capacity (CUPRAC) assays. In addition, this study examined the inhibitory properties of enzymes, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), tyrosinase, alpha-amylase, and alpha-glucosidase. Compared to individual treatments, the combination of GA + HA showed stronger antioxidant abilities in free radical scavenging and reducing power assays in root samples. Moreover, this combination positively influenced the inhibitory effects of root samples on AChE and BChE and the tyrosinase inhibitory effect of leaf samples. Concerning Pearson’s correlations, antioxidant and enzyme inhibition activities were related to phenolic compounds, and lignans in particular correlated with radical scavenging activities. Overall, the tested elicitors could offer promising insights for enhancing the functional properties of lettuce in agricultural treatments.