Oral hygiene practice is known for its significant importance in lowering dental caries risk. To assess the risk factors affecting dental caries, including gender and oral hygiene practices such as regular tooth brushing, use of fluoridated toothpaste, and use of mouthwash. This comparative cross-sectional study included 300 participants visiting a dental clinic in Jeddah, Saudi Arabia. A questionnaire was used to assess demographic data and oral hygiene habits. Tests were conducted to measure salivary secretion rates, buffering capacities, lactobacilli levels, Streptococcus mutans (MS) counts, plaque indices, and erosion scores. Gender was associated with buffering capacity, MS count, plaque index, and erosion scores. High buffering capacity and MS counts were significantly more common among boys than girls. Moreover, the mean plaque index was higher among boys than girls. Regular tooth brushing and mouthwash use were associated with salivary secretion rates. High lactobacilli were more common among those using non-fluoridated toothpaste than those using fluoridated toothpaste. Oral hygiene habits can affect dental caries risk and therefore recommendations are to implement oral hygiene health education programs to reduce dental caries rates in adolescents.