Two novel methods were developed to extract and quantify 56 pesticides in surface waters, considering their content in both dissolved aqueous phase (DAP) and suspended particulate matter (SPM) fractions. These procedures were applied to coastal samples taken seasonally during 2012-2013, from three strategic sampling sites along the Ria Formosa Lagoon (south of Portugal). Briefly, 500 mL of water samples were filtrated, separating both fractions. The DAP fraction was extracted and pre-concentrated by solid-phase extraction (SPE), while the SPM was extracted using ultrasonic extraction technique (USE). Both fractions were then analyzed, and the pesticides were quantified and identified, within 35 min, by gas chromatography (GC) coupled to mass spectrometry (GC-MS and GC-MS/MS), respectively. The extraction of pesticides from the SPM fraction showed average recoveries of 102%, detection limits below 2.2 ng/L, and quantification limits ranging from 0.3 to 6.6 ng/L. Considering the real water samples, 73% of the selected pesticides were quantified in both DAP and SPM fractions (ΣDAP+SPM 2.3 μg/L) and their maximum levels were measured in autumn and winter. By category, the global loads of fungicides, herbicides, and insecticides were ≈407, ≈323, and ≈1.6 μg/L, respectively. Thirty-one percent of the quantified pesticides exceeded the European directives levels (2008/105/EC and 98/83/EC). From the total loads, the SPM fraction contribution was 32%, showing the importance of measuring pesticides in that fraction. The water physicochemical parameters revealed that the total nitrogen amounts were very high relatively to the legal required values, mainly close to the city of Faro (2.6 mg/L). In light of the above, measures are in need to meet European directives and protect both fauna and humans that use this area for leisure.