Implementing nanofertilizers in cultivation to enhance food security is important and gaining great significance, as they have good properties to improve plant production, phytochemicals, and nutrient efficiency and thereby meet the demands of the increasing world population for food. This work demonstrated the impact of calcium carbonate nanoparticles (Ca-NPs) and Ca bulk at three concentrations (0, 100, and 200 mg L−1) on growth, productivity, photosynthetic pigments, phytochemical content, antioxidant activity enzymes, minerals, toxicity, and genomic DNA of lettuce plants. In this regard, Ca-NPs at a concentration of 200 mg L−1 reinforced the vegetative growth characteristics of lettuce plants, increasing head length by 15.7 and 19.2%, head diameter by 20.3 and 19.9%, head fresh weight by 54.4 and 52.9%, and production per hectare by 54.7 and 52.8% as compared to the control during the two growing seasons. Furthermore, the percentages of total chlorophyll (62.6 and 59.5%), carotenoids (48.4 and 56.5%), total phenolics (63.6 and 65.7%), total indoles (39.4 and 36.4%), vitamin C (39.7 and 39.6%), antioxidant activity (57.8 and 53.7%), nitrogen (70.5 and 67.5%), phosphorus (120 and 110.5%), potassium (33.0 and 33.2%), and calcium (67.14 and 63.2%) were also increased compared with the control during two consecutive growing seasons. Additionally, Ca-NPs and Ca bulk had an impact on the plants’ genomic DNA compared to the control. In addition, lettuce plants treated with Ca-NPs were proven to be nontoxic and safe for humans by using the Microtox 500 analyzer.