A second half Joe Adams strike won the match for the young Bees in a game where they showed promise throughout and claimed a deserved victory on the balance of play.
It was a B Team starting XI which also included three First Team players in Luka Racic, Myles Peart-Harris and Dom Thompson, whilst Jan Žambůrek was named on the bench as he stepped up his return from injury.
It was a match where the young Bees started brightly in possession with the likes of Paris Maghoma and Peart-Harris looking to dictate in the middle of the pitch.
The ball found the net early on but for the offside flag which ruled out a Nathan Young-Coombes effort which followed some fantastic and powerful play from Daniel Oyegoke as he burst forward from the right.
Oyegoke actually had the ball in the net himself later but Brentford B were once again denied by the offside flag.
Young-Coombes was also in action at the other end in the first period as he headed away a Tyrone Barnett header that looked destined for the top corner as the youngster somehow managed to glance the ball away and over the bar.
Both sides had a number of free-kicks and they continued to challenge each other aerially going towards the break but a good moment did fall for The Spitfires when Michael Kelly got on the end of an Eastleigh breakaway but Ben Winterbottom saved well to his left.
Neil MacFarlane made changes at the break with Žambůrek replacing the impressive Maghoma and Matt Cox came on in goal for the side.
It was a half where The Bees continued to control much of the action and the winning goal came around 15 minutes from time where the Bees' press, which caused havoc for much of the night, paid off as the pressure saw a loose ball fall to Adams, who held off his man, before rounding the keeper and firing home with his left foot.
Credit to Brentford B who matched their opponents in the air and they defended confidently throughout the final minutes as they limited their opponents to very little in terms of clear chances as Matthew Cox saw a couple of efforts from distance go wide of his goal.
Eastleigh: McDonnell, Hare, Kelly, Miley, Boyce, Pritchard, Barnett, Low, House, Maghoma, Whelan
Subs: Broadbent, Smart, Smith, Hesketh, Blair, Hill, Bragg, Torniainen
Brentford B: Winterbottom, (Cox, 45) Oyegoke, Racic, Hockenhull (Russell, 70) Thompson, Maghoma (Žambůrek, 45) Peart-Harris Haygarth (Trevitt, 70) Brook (Jefferies, 82) Young-Coombes (Hercules, 82) Adams (Trialist, 82)