Lettuce is one of the most consumed leafy greens. Depending on the variety, it is ready for harvesting 40–80 days after sowing, and therefore several growth cycles can be concluded within a growing season. Due to its high market standards, lettuce may require intensive management. This study implemented a critical period of weed interference (CPWI) trial to understand at which moment of the cropping cycle weeds can be tolerated without impacting crop yield to decrease the time needed for weeding and assess the potential support weeds can give to biological pest control in lettuce. Treatments represented two gradients of weed intensity: (1) increasingly weed-free, and (2) increasingly weedy. Dose–response curves were produced to find the CPWI based on lettuce relative yield. RLQ analysis was used to explore the relationships between weeding regime and weed functional traits for biological pest control. Yield was above the 5% acceptable yield loss threshold in all plots kept weed-free for 20 days or more, indicating a necessary weed-free period of 20 days from transplanting. However, the support of beneficial insects was not guaranteed at the end of the necessary weed-free period. We suggest that it is possible to limit intense weed management to the beginning of the growing season, reducing the cost of plastic mulches and increasing on-farm biodiversity, but field margins could be better suited to deliver conservation biological control in short-term crops where this service is of primary interest.