Phenylethanoid glycosides (PhGs) are promising natural products for the treatment of chronic diseases because of their wide range of biological activities. Biotic stress as pathogen attack (fungi) may stimulate the synthesis of PhGs through activation of phenylalanine ammonia-lyase (PAL). Castilleja tenuiflora Benth. (Orobanchaceae) in vitro cultures are alternative sources of PhGs, however in that conditions, cultures have a diminished synthesis of these compounds. To increase the yields of PhGs, the identification of factors affecting their biosynthesis is required. Here, we show that elicitation with cell wall oligosaccharides (CWOs) from Fusarium oxysporum f. sp. lycopersici race 3 (Hyphomycetes) stimulates biosynthesis of PhGs by increasing the activity of PAL. Upon elicitation with CWOs (13 μg/mL) the production of PhGs was enhanced by 5-fold compared with untreated control. The maximum PAL activity in shoots cultured under CWOs elicitation were also increased. Elicitation did not affect the shoot growth (length and biomass) but induced chlorosis, and delayed root formation of C. tenuiflora shoots. Our results demonstrate that elicitation with CWOs increases PhGs biosynthesis in C. tenuiflora shoot culture.